Stress response and adaptation is the basic principle for microorganisms to survive in their natural habitats. Whether they are living in host organisms or directly exposed to nature, they are all equipped with a large number of defense systems against stresses. This proposal is focused on two major stress- response systems in Escherichia coli. First, the molecular mechanisms of stress response through transmembrane histidine kinases, and secondly, the molecular mechanisms of cold-shock response and adaptation. The understanding of these two major stress response systems in E. coli, one through membrane receptors and the other by direct biophysical effects of temperature changes on cellular components will have important implications in studying stress responses in wide varieties of living organisms. We have recently determined the first ever three-dimensional solution structure of the histidine kinase domain of EnvZ, an osmosensor that regulates porin gene expression (ompF and ompC). We also demonstrated that dimerization of histidine kinases is obligatory for its biological function. In this proposal, we attempt to fully decipher the molecular mechanism of (a) the kinase (autophosphorylation and OmpR kinase) and phosphatase (phospho- OmpR dephosphorylation) activities; (b) the transduction of signal across the membrane of ligand binding that regulates histidine kinase function; (c) transcriptional activation and repression of the two porin genes through phospho-OmpR. In the cold-shock project, we have identified CspA, a major cold-shock protein functioning as an RNA chaperone, and 8 other CspA homologues, CspB to CspI. We will continue to elucidate the roles of individual Csp proteins in this nine-membered family. In particular, we will take advantage of a quadruple deletion mutant (DcspA DcspB DcspG DcspE) of E. coli, which is cold- sensitive. This quadruple mutant will allow us to characterize the roles of individual Csp proteins and other functionally related proteins by complementation experiments. We will also continue to investigate the regulatory mechanisms of the cspA expression at low temperature at the level of transcription, mRNA stability and translation initiation. The proposed research will provide important insight into the fundamental principles of stress response and adaptation in prokaryotes.